http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–8 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.962058

ORIGINAL ARTICLE

Anti-hyperlipidemic effect of methanol bark extract of Terminalia chebula in male albino Wistar rats Murali Mohan Reddy1*, Jackson Dhas Devavaram2*, Jebasingh Dhas3, Ernest Adeghate4, and Bright Starling Emerald4

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Department of Pharmacology, Mohamed Sathak A.J. College of Pharmacy, Chennai, Tamil Nadu, India, 2Padmavathi College of Pharmacy, Dharmapuri, Tamil Nadu, India, 3Saastra College of Pharmaceutical Education & Research, Nellore, Andhra Pradesh, India, and 4Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, UAE Abstract

Keywords

Context: Hyperlipidemia is known to be a major risk factor for the development of cardiovascular diseases (CVDs) which include atherosclerosis, coronary heart disease, and stroke. Although there are a large number of anti-hyperlipidemic drugs available, unfortunately, they all have side effects. Objective: Terminalia chebula Retz. (Combretaceae) is a plant used to treat cardiac disorders in the traditional Ayurveda medicine in India. The objective of this study was to assess the antihyperlipidemic properties of a methanol (MeOH) bark extract of T. chebula. Materials and methods: Acute toxicity studies were performed according to the Organisation for Economic Cooperation and Development (OECD) guideline no. 423 using various doses (5, 50, 300, and 2000 mg/kg) of T. chebula bark. Anti-hyperlipidemic effect of MeOH bark extract of T. chebula at doses of 200, 400, and 600 mg/kg and fasting glucose levels after treatment with MeOH bark extract of T. chebula at doses of 200, 400, and 600 mg/kg were analyzed using commercially available kits. Results: Acute toxicity studies did not show any morbidity and mortality at various doses. The MeOH extract of T. chebula bark at doses of 200, 400, and 600 mg/kg significantly lowered serum cholesterol and triglyceride levels. Moreover, the extract of T. chebula and the positive control atorvastatin-treated groups of animals showed a significant increase in the serum highdensity lipoprotein (HDL) cholesterol levels in diet-induced hypercholesterolemic animals. Conclusion: The overall results confirm that the bark extract of T. chebula possesses significant anti-hyperlipidemic activity.

Acute toxicity study, anti-hyperlipidemic property, MeOH bark extract, statins

Introduction Cardiovascular diseases (CVDs) including coronary heart disease and atherosclerosis are the leading causes of death all over the world (Saravanan et al., 2007). According to the World Health Organization (WHO), by the year 2030, more than 23 million people will die annually from CVDs (Misra & Vikram, 2004). High plasma levels of cholesterol along with the generation of reactive oxygen species (ROS) plays a key role in the development of coronary artery disease and atherosclerosis (Visavadiya & Narasimhacharya, 2005). Several factors such as diet high in saturated fatty acids and cholesterol, family history, age, disease conditions like hypertension and lifestyle play a significant role in the etiology of CVDs. However, the high levels of triglycerides (TG) and cholesterols, in particular, total cholesterol (TC), *These authors contributed equally to this work. Correspondence: Dr. Bright Starling Emerald, Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, Tawam Medical Campus, PO Box 17666, Al Ain, United Arab Emirates. E-mail: [email protected]

History Received 17 June 2014 Revised 3 August 2014 Accepted 29 August 2014 Published online 27 January 2015

and low density lipoproteins (LD), are mainly responsible for the onset of coronary heart disease (Stauffer et al., 2013). The American Heart Association also confirms that the primary risk factors associated with atherosclerosis are elevated levels of cholesterol and triglyceride in the blood. A 20% reduction in blood cholesterol level can decrease the incidence of coronary heart disease by 31% and its mortality, by as much as 33% (Marzyieh et al., 2007). Therefore, treatment of hyperlipidemia is one of the major therapeutic approaches towards decelerating the process of atherogenesis (Moss & Dajani, 1971). Different lipid-lowering drugs such as statins, 3-hydroxy3-methyl-glutaryl-CoA reductase (HMG-Co-A reductase) inhibitors, fibric acid derivatives, bile acid sequestrants, cholesterol absorption inhibitors, and nicotinic acid derivatives are used to treat hyperlipidemia. However, one factor which still needs to be considered with the use of these drugs is their adverse effects (Mahley & Bersot, 2006; Malloy & Kane, 2007; Rang et al., 2007; Talbert, 2008), which includes joint and muscular pains, flushing, and many others. Traditionally, plants are the oldest known natural sources of

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medicines with little or no side effects, making them highly useful, and are continually being explored and exploited. As such, many plant species have been used in the traditional Indian Ayurvedic medicine to treat hypelipidemia. For example, extracts of Rhinacanthus nasutus (Acanthaceae) (Desu & Saileela, 2013), Amaranthus spp. (Amaranthaceae) (Girija & Lakshman, 2011), Gmelina arborea (Verbanaceae) (Punitha et al., 2012), Ananas comosus (Bromeliaceae) (Babu et al., 2012), Sphaeranthus indicus (Asteraceae) (Pande & Dubey, 2009), Adenanthera pavonina (Fabaceae) (Das et al., 2011), and Cinnamomum tamala (Lauraceae) (Dhulasavant et al., 2011) possess promising anti-hyperlipidemic activity. Further, search for other existing natural sources of medicines, such as Terminalia chebula Retz. (Combretaceae), with better efficacy and safety cannot be overemphasized. Terminalia chebula is a flowering evergreen tree which is indigenous to many Asian (especially India and Pakistan) and African countries. Fruits, stem, bark, and leaves of this plant are all used as an antioxidant, anti-inflammatory, anti-cancer, hepatoprotectant, and cardiotonic agent. It is confirmed that the methanol extract of T. chebula possesses antibacterial (Lee et al., 2011), free radical scavenging (Elias et al., 2011), and cardiotonic (Babu et al., 2012) activities. An earlier study based on a combination of Gaumutra extract along with the aqueous extract of T. chebula fruit has revealed the presence of an anti-hyperlipidemic activity (Israni et al., 2010). However, the true potential of the anti-hyperlipidemic activity of T. chebula extract (especially the bark) still warrants further investigation. Hence, in the present study, we have assessed the anti-hyperlipidemic properties of the methanol bark extract of T. chebula.

Materials and methods Collection of plant materials and authentication Bark samples of T. chebula were collected from the Chittoor district of Andhra Pradesh, India, during the month of January 2013, and was identified and authenticated by Dr. K. Madhava Chetty, Department of Botany, Sri Venkateswara University, Tirupathi, India. Preparation of MeOH bark extract of T. chebula Dried powdered bark (250 g) of T. chebula was subjected to batch extraction in a Soxhlet apparatus as described in Desu and Saileela (2013). The powdered material of the bark was packed in a Soxhlet extractor (a round-bottom flask) for extraction with 70% methanol (MeOH) at a temperature within a range of 70–75  C, for the experiments. The MeOH extract was then cooled, strained, and filtered. After filtration, the extract was evaporated to dryness by slow heating and continuous stirring in a water bath until the volume was one-fourth of its original volume. The residue was collected and stored in a refrigerator for further use.

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Research & Administration Pvt. Ltd., Chennai, India. The animals were maintained in a well-ventilated room with a 12:12 h light/dark cycle in polypropylene cages, with standard room temperature (25 ± 1  C) and humidity of 44–50%. They were fed with normal chow (commercial pellets) and water ad libitum. All animals were acclimatized to laboratory conditions for a week prior to the experimentation. Thirty-six animals were divided into six groups of six animals in each group, as shown in the following: Group I: Control rats: which were fed with normal diet and water. Group II: Negative control: treated with high-fat diet per os (p.o.) for a period of 30 d. Group III: Positive control: treated with atorvastatin (10 mg/ kg p.o.) and high-fat diet p.o. for a period of 30 d. Group IV: Extract treated rats: treated with MeOH extract of T. chebula bark 200 mg/kg p.o. and high-fat diet p.o. for a period of 30 d. Group V: Extract treated rats: treated with MeOH extract of T. chebula bark 400 mg/kg p.o. and high-fat diet p.o. for a period of 30 d. Group VI: Extract treated rats: treated with MeOH extract of T. chebula bark 600 mg/kg p.o. and high-fat diet p.o. for a period of 30 d. The animals were fasted overnight before experimentation, but allowed free access to drinking water. All experiments were performed in the morning to maintain uniformity with the experiments. Experiments were carried out in accordance with the Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guide lines (769/2011/ CPCSEA and 130/SICRA/IAEC). Determination of ash content The ash content of powdered drug was established by taking the residue remaining after incineration as described in Indian Pharmacopoeia (1985). Total ash was assessed by making a fine layer with 2 g of extract in a silica crucible and incinerating at 450  C to remove carbon. The crucible was then cooled, weighed, and the percentage of total ash was calculated with reference to the air-dried drug. Determination of water soluble ash Water-soluble ash is the part of the total ash content which is soluble in water. To estimate the water soluble ash, the ash was boiled as mentioned before for 5 min with 25 ml of water. The insoluble matter was collected in a Gooch crucible, washed with hot water, and ignited for 15 min at 450  C. The weight of the insoluble matter was subtracted from the weight of the ash and the difference of weight implied the water soluble ash. Determination of acid insoluble ash

Animals Three-month-old male albino Wistar rats weighing 150– 200 g were obtained from the Sigma Institute of Clinical

The total ash obtained was boiled with 25 ml of HCl acid for 5 min. The percentage of acid insoluble ash was calculated with reference to the air-dried drug.

Anti-hyperlipidemic effect of Terminalia chebula

DOI: 10.3109/13880209.2014.962058

Extractive value: ethanol-soluble extract Five grams of T. chebula bark extract was ground into coarse powder, macerated with 100 ml of 95% ethanol in a closed flask for 24 h with intermittent shaking during the first 6 h and filtered. The 25 ml of the filtrate was evaporated to dryness at 105  C and weighed. The percentage of ethanol soluble extract was calculated with reference to the air-dried drug. Water soluble extract

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Another 5 g of T. chebula bark was made into coarse powder and macerated with 50 ml of water at 80  C. After cooling, 2 g of kieselguhr was added and filtered. The 5 ml of the filtrate was evaporated on a water bath, and the residue was weighed. The percentage of water-soluble extract was calculated using the air-dried drug as reference.

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because it contains high saturated fat which enhances the lipid profile in rats (Israni et al., 2010). Histopathological analysis of the liver At the beginning and end of the treatment period, the weights from animals of all groups were taken. On day one, six animals which were fasted overnight were sacrificed and serum and liver collected. Similarly, animals from the different groups were fasted at the end of the experiment, and then sacrificed before their serum and liver tissue samples were collected. Liver tissues were washed, fixed, dehydrated, and processed for light microscopy according to established methods (Adeghate et al., 2001; Jebasingh et al., 2014). The, 5 mm thick tissue sections were prepared and stained with hemotoxylin and eosin and examined by light microscopy.

Loss on drying Loss on drying is the measure of loss in percentage w/w resulting from the loss of water and volatile matter of any kind. A glass-stoppered, deep-weighing bottle was weighed accurately and a definite quantity of the sample was transferred to the bottle covered and weighed. The sample was dried in a drying chamber, cooled at room temperature in a desiccator, and weighed again. Acute toxicity study Acute oral toxicity of MeOH bark extract of T. chebula was carried out according to Organization for Economic Cooperation and Development (OECD) guideline number 423. The method allows judgment with regard to classifying the test material to one of a series of toxicity classes defined by fixed LD50 cut-off values. In this method, the material was examined using a stepwise analysis, where each step used three animals of a single gender normally females. Based on the absence or presence of agent-related mortality of the animals at the given dose, the next phase will be decided, i.e., no additional testing is required, dosing of three more animals with the same dose is needed or, dosing of three additional animals at the next higher or the next lower dose level is required. Healthy, non-pregnant 8–12 weeks old female rats weighing around 150–200 g, acclimatized to the laboratory conditions for a week were used. The levels of used dose falls within the following fixed doses: 5, 50, 300, and 2000 mg/kg body weight. The animals were observed continuously for 2 h for (i) behavioral profile: restlessness, spontaneous irritability, and fearfulness; (ii) neurological profile: spontaneous reactivity, touch response, and pain response; and (iii) autonomic profile: defecation and urination. After a period of 24 h and 72 h, the animals were observed for any toxic signs, lethality, or death. Composition of high-fat diet High-cholesterol diet was prepared by homogenous mixing of cholesterol 2% w/w, sodium cholate 1% w/w, and coconut oil 2.5% w/w with powdered standard rat chow (Ghosh et al., 2010; Hassarajani et al., 2007). Coconut oil was selected

Measurement of glucose and lipid profile Total cholesterol (TC), triglyceride (TG), and high-density lipoprotein (HDL) were measured using PRIETEST Diagnostic Kits, ROBONIK, Thane, India (Product codes PCHO/0612/01; PTRIG/011/01, and PHDL PPT/1211/01). Low-density lipoprotein (LDL) was measured using Friedewann’s equation as per the manufacturer’s instruction (PRIETEST Diagnostic Kits, ROBONIK, Thane, India). Very low-density lipoprotein (VLDL) was calculated by subtracting HDL and LDL form TC. Blood glucose levels were measured from the tail vein of rats using a LifeScan (Lifescan Inc., Milpitas, CA) Glucometer after overnight (12 h) fasting. Statistical analysis Data were recorded as mean ± SEM. The statistical significance of differences between the groups was determined by analysis of variance (ANOVA), followed by Dunnet’s test for multiple comparisons among the groups. Differences of p50.05 were considered statistically significant.

Results Analysis of the physicochemical constants of the MeOH bark extract of T. chebula The MeOH bark extract of T. chebula was subjected to physicochemical analysis and the constants such as the total ash, acid soluble ash, water soluble ash, loss on drying were estimated as per the procedures mentioned in Indian pharmacopoeia, 1985. The total extract value was 17.9% (w/w) with a 9.25% loss of drying. The total ash value was 3.42% (w/w), with 2.63% (w/w) acid insoluble ash and 51.27% (w/w) watersoluble ash (Table 1). Acute toxicity study Acute toxicity studies of the MeOH extract of T. chebula bark were carried out as described in the ‘‘Materials and methods’’ section. No mortality, morphological, or significant behavioral changes were observed even at 2000 mg/kg dose within 48 h. As a result, doses of 200 mg/kg and 400 mg/kg and 600 mg/kg of body weights were chosen for the present study.

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Effects of T. chebula MeOH bark extract on body and liver weights Body and liver weights were analyzed in groups I–VI animals on days 1 and 30. It was seen that the body weight was not significantly different between the groups on day 1. However, on day 30, there was a significant difference in the weight gain between the normal commercial chow-fed control group I animals compared with that of the high-fat diet-fed group II animals where the weight increased significantly in the group II animals (p50.001). The positive control group III animals which were treated with atorvastatin gained less weight than the high-fat diet-fed group II animals (p50.001). There was no significant difference in weight gain between 200 mg/kg MeOH extract of T. chebula dose group IV animals and the high-fat diet-fed group II animals. The 400 mg/kg and 600 mg/kg MeOH extract of T. chebula-treated group V and VI animals also showed less weight gain when compared with

Table 1. The results of the quantitative analysis of physiochemical constants, the total extractive value, total ash, acid in soluble ash, water soluble ash, and loss on drying of MeOH bark extract of T. chebula. Parameters Extractive value Loss on drying Total ash Acid in soluble ash Water soluble ash

Figure 1. The effect of MeOH bark extract of T. chebula on body and liver weights. Comparison of Wistar rats at days 1 and 30. Group II versus groups I, III, IV, V, and VI. Significant difference, *p50.05, **p50.01, and ***p50.001. Group I – vehicle control; Group II – negative control (high-fat diet-fed animals); Group III – positive control (treated with atorvastatin and high-fat diet); Group IV – MeOH bark extract of T. chebula 200 mg/kg and high-fat diet; Group V – MeOH bark extract of T. chebula 400 mg/kg and high-fat diet; Group VI – MeOH bark extract of T. chebula 600 mg/kg and high-fat diet. Values are expressed as mean ± SEM from six male animals in each group.

Results 17.9% w/w 9.25% 3.42% 2.63% 51.27%

the high-fat diet-fed group II animals (p50.01 and p50.001) as presented in Figure 1(A). The liver weight was not significantly different between the groups on day 1. On day 30, there was a significant difference in the liver weights between the normal chow-fed control group I animals compared with that of the high-fat diet-fed group II animals, where the weight increased significantly (p50.001). The weight gain of the liver in the positive control group III animals which was treated with the drug atorvastatin was significantly lower when compared with the high-fat diet-fed group II animals on day 30 (p50.001). The weight gain of the livers in different MeOH extracts of T. chebula-treated animal groups IV, V and VI (200 mg/kg, 400 mg/kg, and 600 mg/kg) were also significantly lower when compared with the high-fat diet-fed group II animals (p50.001) (see Figure 1B). Effect of T. chebula MeOH bark extract on serum lipid profile and blood glucose In comparison with the normal chow-fed control group I animals, the levels of serum total cholesterol, triglyceride, LDL, and VLDL increased significantly in the high-fat dietfed group II animals (p50.001). At the same time, the HDL cholesterol level showed a significant decrease in the high-fat diet-fed group II animals when compared with the normal chow-fed control group I animals (p50.001). The positive control group III animals which were treated with atorvastatin showed a significant decrease in their serum total cholesterol, triglyceride, LDL, and VLDL levels when compared with the high-fat diet-fed group II animals (p50.001). In comparison

Anti-hyperlipidemic effect of Terminalia chebula

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DOI: 10.3109/13880209.2014.962058

with the high-fat diet-fed group II animals, the 200 mg/kg MeOH extract of T. chebula dose group IV animals showed significant decrease serum total cholesterol, triglyceride level (p50.001), and a moderate but significant decrease in their serum LDL and VLDL levels when compared with the highfat diet-fed group II animals (p50.05). The 400 mg/kg MeOH and 600 mg/kg MeOH extract of T. chebula dose group V and VI animals showed a significant decrease in their serum total cholesterol, triglyceride, LDL and VLDL levels when compared with the high-fat diet-fed group II animals (p50.001). The HDL cholesterol level also showed a significant increase in the positive control group III animals, 400 mg/kg MeOH and 600 mg/kg MeOH extract T. chebula dose group V and VI animals when compared with the highfat diet-fed group II animals (p50.001), where it was moderate but significant (p50.05) in the 200 mg/kg MeOH extract of T. chebula dose group IV animals when compared with the high-fat diet-fed group II animals (Table 2). The fasting blood glucose was also found to be in the diabetic range in the high-fat diet-fed group II animals when compared with the normal chow-fed control group I animals (138.43 ± 0.800 versus 74.58 ± 0.775 mg/dl). The positive control group III animals which were treated with atorvastatin showed a significant decrease in their fasting blood glucose level (93.98 ± 0.511 mg/dl). In comparison with the high-fat diet-fed group II animals, the 200 mg/kg, 400 mg/kg MeOH, and 600 mg/kg MeOH extract of T. chebula dose group IV, V, and VI animals showed a significant decrease in their fasting blood glucose levels (120.95 ± 1.097, 116.62 ± 0.582, and 97.13 ± 0.329 mg/dl, p50.05), respectively, which are within the normal blood glucose levels (Table 2). In comparison with the normal chow-fed control group I animals, the serum total cholesterol/HDL ratio, antherogenic index (TC-HDL/HDL), and the LDL/HDL ratio increased significantly in the high-fat diet-fed group II animals (p50.001). The positive control group III animals which were treated with atorvastatin showed a significant decrease in the total cholesterol/HDL ratio, antherogenic index, and the LDL/HDL ratio when compared with that of the high-fat dietfed group II animals (p50.001). In comparison with the highfat diet-fed group II animals, the 200 mg/kg, 400 mg/kg MeOH, and 600 mg/kg MeOH extract T. chebula dose group IV, V, and VI animals showed a significant decrease in the

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total cholesterol/HDL ratio, antherogenic index, and the LDL/ HDL ratio (p50.001) (Table 3). Effects of T. chebula MeOH extract on liver histopathology The histopathological analysis of the livers from the high-fat diet-fed group II animals by light microscopy showed dilatations in the sinusoidal capillaries of the liver (Figure 2B, arrow) when compared with the normal chow-fed control group I animals (Figure 2A). The high-fat diet-fed group II animals also showed fatty cytoplasmic-vacuolated hepatocytes and hepatocytes with large cytoplasmic vacuoles (Figure 2B, dotted arrow and asterisk). The positive control group III animals which were treated with atorvastatin showed a significant decrease in the sinusoidal capillary dilatations with negligible cytoplasmic fat deposition and granular degeneration (Figure 2C). In comparison with the high-fat diet-fed group II animals the 200 mg/kg MeOH extract T. chebula dose group IV animals showed moderate decrease in the sinusoidal capillary dilatations, the fat infiltration, and granular degeneration (Figure 2D). The 400 mg/kg MeOH and 600 mg/kg MeOH extract T. chebula dose group V and VI animals showed a significant decrease in the dilatations in the sinusoidal capillaries of the liver, Table 3. Effect of MeOH extract of T. chebula bark on different cardiac risk factors.

Groups

Total cholesterol/HDL ratio

Atherogenic index

LDL/HDL ratio

Group Group Group Group Group Group

2.047 ± 0.043*** 10.28 ± 0.1808 2.07 ± 0.025*** 7.862 ± 0.096*** 5.153 ± 0.069*** 2.717 ± 0.025***

1.050 ± 0.042*** 9.275 ± 0.190 1.074 ± 0.025*** 6.864 ± 0.096*** 4.157 ± 0.068*** 1.719 ± 0.025***

0.71 ± 0.038*** 7.83 ± 0.167 0.74 ± 0.022*** 5.72 ± 0.081*** 3.48 ± 0.062*** 1.29 ± 0.024***

I II III IV V VI

LDL, low-density lipoprotein; HDL, high-density lipoprotein. Group I – vehicle control; Group II – negative control (high-fat diet-fed animals); Group III – positive control (treated with atorvastatin and high-fat diet); Group IV – MeOH bark extract of T. chebula 200 mg/kg and high-fat diet; Group V – MeOH bark extract of T. chebula 400 mg/kg and high-fat diet; Group VI – MeOH bark extract of T. chebula 600 mg/ kg and high-cholesterol diet. Values are expressed as mean ± SEM from six male animals in each group. Significant difference, *p50.05, **p50.01, and ***p50.001.

Table 2. Effect of MeOH bark extract of T. chebula bark on serum total cholesterol, triglyceride, HDL, LDL, VLDL, and glucose level.

Group Group Group Group Group Group Group

I II III IV V VI

Serum total cholesterol (mg/dl)

Serum triglyceride (mg/dl)

HDL cholesterol (mg/dl)

Serum LDL cholesterol (mg/dl)

Serum VLDL cholesterol (mg/dl)

Glucose (mg/dl)

97.05 ± 0.933*** 268.10 ± 2.282 116.81 ± 1.050*** 239.06 ± 0.560** 204.81 ± 1.167** 138.43 ± 1.899***

77.73 ± 0.660*** 186.35 ± 2.810 91.15 ± 0.432*** 171.53 ± 1.382*** 133.3 ± 0.497*** 108.4 ± 1.874***

47.36 ± 0.604*** 26.13 ± 0.452 56.33 ± 0.573*** 30.40 ± 0.380* 39.75 ± 0.395*** 50.90 ± 0.598***

34.06 ± 1.420*** 204.02 ± 2.284 42.25 ± 1.035*** 174.08 ± 0.902* 138.46 ± 1.366*** 65.84 ± 1.472***

15.41 ± 0.193*** 37.33 ± 0.387 18.24 ± 0.083*** 34.30 ± 0.676* 26.37 ± 0.360*** 21.68 ± 0.374***

74.58 ± 0.775 138.43 ± 0.800 93.98 ± 0.511 120.95 ± 1.097 116.62 ± 0.582 97.13 ± 0.329

Group I – vehicle control; Group II – negative control (high-fat diet-fed animals); Group III – positive control (treated with atorvastatin and high-fat diet); Group IV – MeOH bark extract of T. chebula 200 mg/kg and high-fat diet; Group V – MeOH bark extract of T. chebula 400 mg/kg and high-fat diet; Group VI – MeOH bark extract of T. chebula 600 mg/kg and high-fat diet. Values are expressed as mean ± SEM from six male animals in each group. Significant difference, *p50.05, **p50.01, and ***p50.001.

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Figure 2. Effect of MeOH bark extract of T. chebula on liver histopathology. Representative photographs of the liver sections from the different groups. (A) Group I – vehicle control; (B) Group II – negative control (high-fat diet-fed animals) with sinusoidal capillary dilatations (arrow), fatty infiltrated hepatocytes (dotted arrow), and hepatocytes with large cytoplasmic vacuoles (*); (C) Group III – positive control (treated with atorvastatin and high fat diet); (D) Group IV – MeOH bark extract of T. chebula 200 mg/kg and high-fat diet; (E) Group V – MeOH bark extract of T. chebula 400 mg/kg and high-fat diet; (F) Group VI – MeOH bark extract of T. chebula 600 mg/kg and high-fat diet.

cytoplasmic fatty infiltration, and granular degeneration when compared with the high-fat diet-fed group II animals (Figure 2E and F).

Discussion This study evaluated the effect of anti-hyperlipidemic effect of the MeOH bark extract of T. chebula bark on high-fat dietinduced hyperlipidemia in albino Wistar rats. The extract was subjected to estimation of physicochemical constants like ash values and extractive values in order to satisfy the basic pharmacognostic criteria. An acute toxicity study was performed according to OECD guideline no. 423. The absence of lethality at the limit dose of 2000 mg/kg suggests that the extract of T. chebula is safe even at very high concentrations. Metabolic diseases which result from altered balance between energy intake and expenditure are the new epidemic affecting the world today. High-fat diet is a major cause of obesity which is closely linked to a variety of health issues, including coronary heart disease, stroke, high blood pressure, fatty liver disease, diabetes, and certain cancers (Grundy, 2004; Kahn & Flier, 2000). High-fat diet contributes to the generation of free radical metabolites and the development of systemic inflammation and insulin resistance (Grundy, 2004; Kahn & Flier, 2000). An elevated level of cholesterol with increased levels of triglyceride-rich VLDL and

cholesterol-rich LDL in the circulation is a recognized risk factor of coronary artery disease (Lewington et al., 2007; Thompson, 1990). Animal studies have also shown that an increase in the intake of high calorie and high-fat diet will result in a concomitant increase in the amount of fatty acids in the circulation with an increase in the de novo lipogenesis leading to fatty liver which was mimicked successfully by the high-fat diet. This has been effectively attenuated by the extract of T. chebula, which shows the presence of an antihyperlipdemic effect (Marra et al., 2008). Atrovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, belongs to the class of drugs known as statins. Statins inhibit the synthesis of mevalonate, a rate-limiting enzyme in cholesterol biosynthesis. This results in the reduction of the plasma LDL levels with an increase in the hepatic uptake, thereby reducing the risk of CVDs (Bertolini et al., 1997; Scandinavian Simvastatin Survival Study Group, 1994). Statins are used in clinical practice as a potent cholesterol lowering drug and so the use of it as a positive control in our experiments gave us a better comparison for the anti-hyperlipdemic effect of the extract of T. chebula. The rats gained weight differently during the treatment period. There was a significant increase in the weights of group II animals (negative control). The significantly higher weight gain in the high-fat diet groups of rats than in the controls suggests the influence of high-cholesterol diet

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DOI: 10.3109/13880209.2014.962058

(Sethupathy et al., 2002). The atorvastatin-treated group III animals showed the effect of anti-hyperlipidemic activity as the body weight exhibited a significant reduction in weight gain on day 30. Animals treated with 200 mg/kg p.o. MeOH did not show significant decrease in weight gain during the treatment period. However, animals treated with 400 mg/kg and 600 mg/kg MeOH p.o. showed significant decrease in weight gain on day 30 when compared with the high-fat fed group II animals. The weight of liver also increased significantly in the high-fat diet group II compared with normal control group I animals. All the MeOH treated (200, 400, and 600 mg/kg) group IV, V, VI, and standard atorvastatin-treated group III animals showed significant decrease in liver weight gain compared with high-fat diet negative control group II animals. These results together suggest that the MeOH extract of T. chebula bark possesses anti-hyperlipidemic activity against high-cholesterol dietinduced hyperlipidemia in albino Wistar rats. A high-fat diet increases the level of total cholesterol, with the accumulation of lipid-containing particles in the walls of coronary arteries and other major arteries within the body leading to atherosclerosis and obesity (Indulski & Lutz, 1999). An elevated atherogenic index indicates the deposition of foam cells or plaque or fatty infiltration or lipids in heart, coronary arteries, aorta, liver, and kidneys, which in turn possess a higher risk for the development of oxidative damage (Mehta et al., 2003). The LDL/HDL-cholesterol ratio has been described as a valuable tool to evaluate coronary heart disease risks and is increasingly being recognized as a stronger risk predictor of CVDs than each lipid parameter taken alone (Fernandez & Webb, 2008; Momiyama et al., 2012). Changes in ratios have been shown to be better indicators of successful coronary heart disease risk reduction than changes in absolute levels of lipids or lipoproteins. It is also an excellent indicator for the effectiveness of lipid-lowering therapies (Natarajan et al., 2003; Walldius & Jungner, 2005). Treatment with MeOH extract of T. chebula bark showed a significant decrease in the atherogenic index, total cholesterol/HDL ratio, and LDL/HDL ratio further suggesting the anti-hyperlipidemic potential of T. chebula. High-cholesterol diet induced hyperlipidemia is associated with increased levels of lipids in the serum. The results presented in this study indicated that the MeOH extract of T. chebula bark at doses of 200 mg, 400 mg, and 600 mg effectively prevented the high-fat diet induced increase in the levels of total cholesterol, triglycerides, HDL, LDL, and VLDL. Earlier studies have shown that both LDL and VLDL have a positive role in atherogenesis (Diaz et al., 1997; Pedersen, 2001; Parthasarathy et al., 1989). A rise in LDL leads to the deposition of cholesterol in arteries and aorta and, therefore, it is a risk factor for the development of coronary heart disease (Libby, 1995; Pedersen, 2001). Low level of HDL is associated with high risk of coronary artery disease (Boden & Pearson, 2000). Along with the increase in the levels of serum total cholesterol, triglycerides, LDL, and VLDL, there was also a significant reduction in HDL level in high-fat diet-fed rats. The HDL is synthesized mainly in intestine and liver. The HDL is considered to be a beneficial lipoprotein as it has an inhibitory effect in the pathogenesis of atherosclerosis. A low level of HDL is associated with high

Anti-hyperlipidemic effect of Terminalia chebula

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risk of coronary artery disease (Boden & Pearson, 2000). Treatment with MeOH bark extract of T. chebula at doses of 200 mg, 400 mg, and 600 mg also effectively increased the level of HDL cholesterol. A significant increase in the levels of glucose was observed in high-cholesterol diet-treated group II animals. While 200 mg/kg MeOH bark extract of T. chebula-treated group IV animals showed a slight reduction in serum glucose levels, treatment with 400 mg/kg and 600 mg/kg of MeOH bark extract of T. chebula showed a marked reduction in glucose levels when compared with high-fat diet-treated animals. Liver is an organ of importance in both lipid and glucose metabolism. It plays a major role as the site for the storage and release of carbohydrates and in the synthesis of fatty acids. It has been proposed that the changes in lipid and glucose metabolism in the liver, with impaired catabolism contributing to obesity and insulin resistance, leading to diabetes, liver disease, and metabolic syndrome (Parekh & Anania, 2007; Wakil & Abu-Elheiga, 2009). Histopathological examination of liver in the normal group showed normal architecture which is altered in the high-fat diet-fed group II animals with sinusoidal capillary dilatations, fatty infiltrated hepatocytes, and hepatocytes with large cytoplasmic vacuoles. Treatment with MeOH bark extract of T. chebula at different doses such as 200, 400, and 600 mg/kg showed the architecture of liver returning to normality at various levels confirming the anti-hyperlipidemic action of the plant extract. Lowering the cholesterol levels significantly reduce the risks of heart attacks, strokes, high blood pressure, fatty liver disease, diabetes, and certain cancers. Although the extract of T. chebula at different doses showed the presence of antihyperlipidemic activity, understanding the molecular mechanism of action and the phyto-constituents which are responsible for the observed anti-hyperlipidemic activity may help in devising better intervention strategies for high-fat-induced metabolic diseases.

Conclusion The MeOH bark extract of T. chebula showed dose-dependent anti-hyperlipidemic action against high-fat diet-induced hyperlipidemia in rats. The acute toxicity study indicated that these extracts are devoid of major toxic effects. Administration of the extract at a dose of 600 mg/kg produced significant anti-hyperlipidemic activity in high-cholesterol diet-induced hyperlipidemic rats. Besides this, there was a significant reduction in blood glucose level as well. Further isolation, characterization, and purification of the active constituents and establishment of the molecular mechanism(s) of the action of the extract of T. chebula may establish it as an alternate therapy in the treatment of hyperlipidemia.

Declaration of interest The authors declare that there are no conflicts of interests.

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Anti-hyperlipidemic effect of methanol bark extract of Terminalia chebula in male albino Wistar rats.

Hyperlipidemia is known to be a major risk factor for the development of cardiovascular diseases (CVDs) which include atherosclerosis, coronary heart ...
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